In vitro testing of Nd:YAG laser processed calcium phosphate coatings.

Nd:YAG laser cladding is a new method for deposition of a calcium phosphate onto metallic surfaces of interest in implantology. The aim of this study was to compare the biologic response of MG-63 human osteoblast-like cells grown on Ti-6Al-4V substrates coated with a calcium phosphate layer applied using different methods: plasma spraying as reference material and Nd:YAG laser cladding as test material. Tissue culture polystyrene was used as negative control. The Nd:YAG laser clad material showed a behaviour similar to the reference material, plasma spray, respective to cell morphology (SEM observations), cell proliferation (AlamarBlue assay) and cytotoxicity of extracts (MTT assay). Proliferation, as measured by the AlamarBlue assay, showed little difference in the metabolic activity of the cells on the materials over an 18 day culture period. There were no significant differences in the cellular growth response on the test material when compared to the ones exhibited by the reference material. In the solvent extraction test all the extracts had some detrimental effect on cellular activity at 100% concentration, although cells incubated in the test material extract showed a proliferation rate similar to that of the reference material. To better understand the scope of these results it should be taken into account that the Nd:YAG clad coating has recently been developed. The fact that its in vitro performance is comparable to that produced by plasma spray, a material commercially available for more than ten years, indicates that this new laser based method could be of commercial interest in the near future.

Effect of the particle size on the micro and nanostructural features of a calcium phosphate cement: a kinetic analysis.

The aim of this work is to investigate the possibility of controlling the final micro and nanostructural features of a calcium phosphate cement by modifying the particle size of the starting powder, and to study the effect of this parameter on the kinetics of the setting reaction. The development of calcium phosphate materials with tailored structures at the micro and nanoscale levels could allow the modulation of some specific responses in biologic phenomena such as protein adsorption and cell adhesion, which strongly depend on the nano-sized roughness of the interface. It is shown that the higher specific surface, produced by the reduction of the particle size of the powder, strongly accelerates the hydrolysis of the alpha-TCP into calcium-deficient hydroxyapatite. The higher degree of supersaturation attained in the solution favours the nucleation of smaller crystals. Thus, by increasing the specific surface of the starting powder in a factor of 5, the size of the precipitated crystals is strongly reduced, and the specific surface of the set cement increases by a factor of 2. The reduction of the particle size produces a substantial decrease of the setting time and accelerates the hardening of the cement without significantly affecting the final strength attained. The mechanical strength achieved by the cement cannot be univocally related to the degree of reaction, without considering the microstructural features.

Calcium phosphate coatings obtained by Nd:YAG laser cladding: physicochemical and biologic properties.

The plasma spray (PS) technique is the most popular method commercially in use to produce calcium phosphate (CaP) coatings to promote fixation and osteointegration of the cementless prosthesis. Nevertheless, PS has some disadvantages, such as the poor coating-to-substrate adhesion, low mechanical strength, and brittleness of the coating. In order to overcome the drawbacks of plasma spraying, we introduce in this work a new method to apply a CaP coating on a Ti alloy using a well-known technique in the metallurgical field: laser surface cladding. The physicochemical characterization of the coatings has been carried out by means of X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). The biologic properties of the coatings have been assessed in vitro with human osteoblast-like MG-63 cells. The overall results of this study affirm that the Nd:YAG laser cladding technique is a promising method in the biomedical field.

The Ca/P range of nanoapatitic calcium phosphate cements.

Nanoapatites are apatites consisting of nanometer size crystals. The commercial calcium phosphate cements set by the precipitation of nanoapatitic calcium phosphates in the range 1.5 < or = Ca/P < 1.8. In this study it is shown that a continuum of nanoapatites can precipitate in the range 0.8 < Ca/P< or = 1.5. In order to be formed these nanoapatites need to incorporate K+ ions. In addition they can incorporate some Na+ ions. Upon immersion in aqueous solutions these nanoapatites loose phosphate, K+ and Na+ so that in an open system they are transformed into calcium deficient hydroxyapatite Ca9(HPO4)(PO4)5OH within about 2 months.

Transforming growth factor-beta1 incorporation in a calcium phosphate bone cement: material properties and release characteristics.

The bone regenerative properties of calcium phosphate cements (CPCs) may be improved by the addition of growth factors, such as recombinant human transforming growth factor-beta1 (rhTGF-beta1). Previously, we showed that rhTGF-beta1 in CPC stimulated the differentiation of preosteoblastic cells from adult rat long bones. The intermixing of rhTGF-beta1 in CPC, which was subsequently applied to rat calvarial defects, enhanced bone growth around the cement and increased the degradation of the cement. However, it is unknown whether the addition of rhTGF-beta1 changes the material properties of CPC and what the characteristics of the release of rhTGF-beta1 from CPC are. Therefore, we determined in this study the release of rhTGF-beta1, in vitro, from the cement pellets as implanted in the rat calvariae. The possible intervening effects of rhTGF-beta1 intermixing on the clinical compliance of CPC were studied through an assessment of its compressive strength and setting time, as well as its crystallinity, calcium-to-phosphorus ratio, porosity, and microscopic structure. We prepared CPC by mixing calcium phosphate powder (58% alpha-tricalcium phosphate, 25% anhydrous dicalcium phosphate, 8.5% calcium carbonate, and 8.5% hydroxyapatite) with a liquid (3 g/mL). The liquid for standard CPC consisted of water with 4% disodium hydrogen phosphate, whereas the liquid for modified CPC was mixed with an equal amount of 4 mM hydrochloride with 0.2% bovine serum albumin. The hydrochloride liquid contained rhTGF-beta1 in different concentrations for the release experiments. Most of the rhTGF-beta1 incorporated in the cement pellets was released within the first 48 h. For all concentrations of intermixed rhTGF-beta1 (100 ng to 2.5 mg/g of CPC), approximately 0.5% was released in the first 4 h, increasing to 1.0% after 48 h. Further release was only about 0.1% from 2 days to 8 weeks. CPC modification slightly increased the initial setting time at 20 degrees C from 2.6 to 5 min but had no effect on the final setting time of CPC at 20 degrees C or the initial and final setting times at 37 degrees C. The compressive strength was increased from 18 MPa in the standard CPC to 28 MPa in the modified CPC only 4 h after mixing. The compressive strength diminished in the modified CPC between 24 h and 8 weeks from 55 to 25 MPa. No other significant change was found with the CPC modification for rhTGF-beta1. X-ray diffraction revealed that standard and modified CPCs changed similarly from the original components, alpha-tricalcium phosphate and anhydrous dicalcium phosphate, into an apatite cement. The calcium-to-phosphorus ratio, as determined with an electron microprobe, did not differ for standard CPC and modified CPC. Standard and modified CPCs became dense and homogeneous structures after 24 h, but the modified CPC contained more crystal plaques than the standard CPC, as observed with scanning electron microscopy (SEM). SEM and back- scattered electron images revealed that after 8 weeks the cements showed equally and uniformly dense structures with microscopic pores (<1 microm). Both CPCs showed fewer crystal plaques at 8 weeks than at 24 h. This study shows that CPC is not severely changed by its modification for rhTGF-beta1. The prolonged setting time of modified cement may affect the clinical handling but is still within acceptable limits. The compressive strength for both standard and modified cements was within the range of thin trabecular bone; therefore, both CPCs can withstand equal mechanical loading. The faster diminishing compressive strength of modified cement from 24 h to 8 weeks likely results in early breakdown and so might be favorable for bone regeneration. Together with the beneficial effects on bone regeneration from the addition of rhTGF-beta1 to CPC, as shown in our previous studies, we conclude that the envisaged applications for CPC in bone defects are upgraded by the intermixing of rhTGF-beta1. Therefore, the combination of CPC and rhTGF-beta1 forms a promising synthetic bone graft.

Alloying of hydroxyapatite onto Ti6Al-4V by high power laser irradiation.

In the biomedical field, the synthetic hydroxyapatite [Ca(10)(PO)(4)(OH)(2)], with similarity to the inorganic component of bone but brittle, has been considered as the appropriate coating on stronger implant materials, such as metallic implants, for presenting a surface which is conductive to bone formation. Many industrial and laboratory techniques were developed to apply hydroxyapatite onto metallic substrates, such as electrophoretic deposition, ion sputtering, hot isostatic pressing, pulsed laser deposition and the only widely used method commercially available: plasma spraying. This work presents a new approach on how to bind calcium phosphate (CaP) to the Ti alloy with a well-known technique in the metallurgical field: laser surface alloying, in order to overcome the drawbacks of plasma spraying. The analysis of the results obtained and the description of the phenomena that take place in the coating process will complete this explorative study.

Evaluation of calcium phosphates and experimental calcium phosphate bone cements using osteogenic cultures.

In this study, rat bone marrow cells (RBM) were used to evaluate two biodegradable calcium phosphate bone cements and bioactive calcium phosphate ceramics. The substances investigated were: two novel calcium phosphate cements, Biocement F and Biocement H, tricalcium phosphate (TCP), surface-modified alpha-tricalcium phosphate [TCP (s)] and a rapid resorbable calcium phosphate ceramic consisting of CaKPO(4) (sample code R5). RBM cells were cultured on disc-shaped test substrates for 14 days. The culture medium was changed daily and also examined for calcium, phosphate, and potassium concentrations. Specimens were evaluated using light microscopy, and morphometry of the cell-covered substrate surface, scanning electron microscopy, and energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. Areas of mineralization were identified by tetracyline labeling. Except for R 5, rat bone-marrow cells attached and grew on all substrate surfaces. Of the different calcium phosphate materials tested, TCP and TCP (s) facilitated osteoblast growth and extracellular matrix elaboration to the highest degree, followed by Biocements H and F. The inhibition of cell growth encountered with R 5 seems to be related to its high phosphate and potassium ion release.

Effect of temperature and immersion on the setting of some calcium phosphate cements.

Calcium phosphate cements based on powders containing alpha-Ca3(PO4)2 and aqueous solutions containing Na2HPO4 as accelerator were used to determine the effects of accelerator concentration, temperature and immersion on the setting time. Increases in accelerator concentration and temperature increased the rate of setting, but immersion had a retarding effect. These results were used to design a method whereby a syringe filled with cement paste can be kept ready for injection of the paste into the implantation site for a long time, whereas setting of the cement paste in the body takes place in a short time.

Addition of cohesion promotors to calcium phosphate cements.

Many calcium phosphate cements (CPC) pastes tend to disintegrate upon early contact with blood or other aqueous (body) fluids, which inhibits the use of these materials for clinical use as for bone repair, reconstruction and augmentation. In studies on CPCs based on tetracalcium phosphate and dicalcium hydrogen phosphate others have suggested to use sodium alginate, cellulose derivatives or chitosan derivatives dissolved in the cement liquid for improving the cohesion of CPC pastes. In this study 10 other organic compounds were shown to act as cohesion promotors in the case of CPCs based on alpha-tertiary calcium phosphate as the main active ingredient.

Incorporation of a controlled-release glass into a calcium phosphate cement.

A so-called controlled-release glass was synthesized occurring in the system CaO-Na2O-P2O5. A certain sieve fraction of this glass was incorporated in a calcium phosphate cement, of which the powder contained alpha-tricalcium phosphate (alpha-TCP), dicalcium phosphate (DCP) and precipitated hydroxyapatite (HA). The glass appeared to retard the cement setting slightly and it reduced considerably the compressive strength after aging in aqueous solutions which were continuously refreshed. Scanning electron microscope (SEM) pictures and X-ray diffraction (XRD) patterns of the samples after 5 weeks of aging showed that the glass was not dissolved but that large brushite crystals were formed. Thereby, aging in CaCl2 solutions resulted in more brushite formation than aging in NaCl solutions. The brushite crystals did not reinforce the cement. Neither was the aged glass-containing cement weaker than it was before the brushite formation right after complete setting. In conclusion, the incorporation of controlled-release glasses into a calcium phosphate cement and subsequent aging in aqueous solutions did not result in the formation of macropores in the cement structure, but that of brushite crystals. This incorporation reduced the compressive strength of the cement considerably.

Production and characterization of new calcium phosphate bone cements in the CaHPO4-alpha-Ca3(PO4)2 system: pH, workability and setting times.

The initial setting properties of calcium phosphate cements in the CaHPO4-alpha-Ca3(PO4)2 (DCP-alpha-TCP) system have been investigated. Interest was focused on the pH, workability, cohesion time and initial and final setting times. The addition of CaCO3 modified the structure of the cement reaction product such that it became more similar to the apatite phase in bone mineral. The addition of 10% w/w of CaCO3 reduced the viscosity of the cement pastes resulting in an increase in initial and final setting times and improved injectability.

Calcium phosphate bone cements for clinical applications. Part I: solution chemistry.

Calcium phosphate cements have been the subject of many studies in the last decade because of their biocompatibility, their capacity to fill bone cavities and their hardening properties; properties which are desirable in a broad range of surgical applications. The setting and hardening of these materials are controlled by dissolution-precipitation chemical reactions at room or body temperature and involve crystalline phase transformations.

Calcium phosphate bone cements for clinical applications. Part II: precipitate formation during setting reactions.

Calcium phosphate bone cements (CPBC) have been of great interest in medicine and dentistry due to their excellent biocompatibility and bone-repair properties. In this article, a review is presented of the scientific literature concerning precipitate formation during setting reactions of CPBCs. Firstly, the available information has been classified according to the intended final product or calcium phosphate formed during setting reactions. Taking the final product into account, a second classification has been made according to the calcium phosphates present in the original powder mixture. This is the most natural classification procedure because it is based on thermodynamic reasons supported by solubility diagrams for the calcium phosphate salts. By understanding the thermodynamics of calcium phosphate salts in an aqueous solution at room or body temperature it is possible to optimize the manufacturing technology involved in the production of CPBCs. Knowledge of the limitations of this thermodynamic approach opens up new possibilities in the search for CPBCs with better in vitro and in vivo properties for clinical applications.

Osteotransductive bone cements.

Calcium phosphate bone cements (CPBCs) are osteotransductive, i.e. after implantation in bone they are transformed into new bone tissue. Furthermore, due to the fact that they are mouldable, their osteointegration is immediate. Their chemistry has been established previously. Some CPBCs contain amorphous calcium phosphate (ACP) and set by a sol-gel transition. The others are crystalline and can give as the reaction product dicalcium phosphate dihydrate (DCPD), calcium-deficient hydroxyapatite (CDHA), carbonated apatite (CA) or hydroxyapatite (HA). Mixed-type gypsum-DCPD cements are also described. In vivo rates of osteotransduction vary as follows: gypsum-DCPD > DCPD > CDHA approximately CA > HA. The osteotransduction of CDHA-type cements may be increased by adding dicalcium phosphate anhydrous (DCP) and/or CaCO3 to the cement powder. CPBCs can be used for healing of bone defects, bone augmentation and bone reconstruction. Incorporation of drugs like antibiotics and bone morphogenetic protein is envisaged. Load-bearing applications are allowed for CHDA-type, CA-type and HA-type CPBCs as they have a higher compressive strength than human trabecular bone (10 MPa).

Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.

Materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications for bone regeneration and for use in skeletal reconstruction and joint replacement. We have studied the growth and interactions of human bone marrow cells on a variety of new cement composites in vitro. These cement materials are composed of calcium-deficient hydroxyapatites, carbonated apatite and amorphous calcium phosphate. Cell proliferation was significantly reduced and cell differentiation increased in the presence of these cements compared with cells cultured on tissue culture plastic. Alkaline phosphatase, one of the markers of the osteoblast phenotype, was dramatically stimulated by 3 of the 4 cements examined between day 4 and day 10, above levels observed following culture of human osteoblasts on plastic alone. Photomicroscopic examination demonstrated growth and close integration of bone marrow cells and 3 of the composites. Longer term marrow cultures (15 day) on the cements confirmed the stimulation of cell differentiation over proliferation. From these studies, enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas, cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro culture system demonstrates the use of human bone marrow cells for the potential evaluation of new biomaterials and the development of a novel carbonated apatite that may be of potential use in orthopaedic implants.

Effects of novel calcium phosphate cements on human bone marrow fibroblastic cells.

The identification and characterization of biocompatible materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications in skeletal reconstruction and joint replacement. In the present study, we have examined the effects of three biocements, biocement H, calcium-deficient apatite; biocement F, apatite + CaHPO(4); biocement D, carbonated apatite + CaHPO(4) + CaCO(3) and an amorphous calcium phosphate (ACP) proposed as implant fixing materials, on the growth, differentiation, and cell surface interaction of human bone marrow fibroblastic cells. These cells are known to be progenitors of osteoblasts, chondroblasts, adipocytes, myoblasts, and reticulocytes. Alkaline phosphatase enzyme activity, a marker of the osteoblast phenotype, was increased by a factor of two- to sixfold on carbonated apatite, one- to sixfold on apatite and three- to 10-fold on calcium-deficient apatite, over levels observed on plastic. Cell proliferation was significantly reduced. Photomicroscopic examination indicated high biocompatibility with close adhesion of the bone marrow fibroblastic cells to composites D, F, and H. Longer term marrow cultures (15 days) confirmed the stimulation of cell differentiation, as assessed by collagen production, over cell proliferation, of cells grown on carbonated apatite. Enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro human bone marrow fibroblast culture system provides a simple and effective method for the evaluation of new biomaterials. The development of these novel cements may be of potential use in orthopedic implants.

The cement setting reaction in the CaHPO4-alpha-Ca3(PO4)2 system: an X-ray diffraction study.

The setting reactions of calcium phosphate cements in the CaHPO4-alpha-Ca3(PO4)2 (DCP-alpha-TCP) system have been investigated. X-ray diffraction (XRD) analyses were performed on DCP-alpha-TCP cement samples of varying calcium to phosphorus (Ca/P) ratios after setting for 24 h in Ringer's solution at 37 degrees C. XRD measurements showed that the intensity of the DCP peaks decreased linearly as the Ca/P ratio of the mixture increased. However, the intensity of the peaks of a new calcium-deficient hydroxyapatite [CDHA; Ca9(HPO4)(PO4)5OH] precipitating phase increased linearly as the Ca/P ratio increased. Alpha-TCP was not detected after 24 h of setting in any sample. A two-phase mixture XRD model was applied to explain the results, and suitable fits were obtained between observed and expected values of the relevant peak heights. The method used for this study also can be applied to studies of the kinetic behavior of other cement systems.

Improvement of the mechanical properties of new calcium phosphate bone cements in the CaHPO4-alpha-Ca3(PO4)2 system: compressive strength and microstructural development.

The hardening properties of calcium phosphate cements in the CaHPO4-alpha-Ca3(PO4)2 (DCP-alpha-TCP) system have been investigated with interest focused on the compressive strength and microstructure development. Previous studies have shown that the addition of CaCO3(CC) leads to a modification of the calcium-deficient apatite structure of the reaction product, which results in a material more similar to the apatite in bone mineral. The addition of 10% w/w of CC to the initial DCP-alpha-TCP powder mixture resulted, with time, in a retardation of the development of compressive strength. However, the optimum compressive strength reached values up to 40% higher than CC-free samples. This retarding effect also has been monitored as a function of the calcium to phosphorus (Ca/P) ratio of the DCP and alpha-TCP mixture, showing the importance of the final cement properties of the relative quantities of the reactants in the mixture.

Limited compliance of some apatitic calcium phosphate bone cements with clinical requirements.

Clinical requirements for calcium phosphate bone cements were formulated in terms of the initial setting time, the final setting time, the cohesion time and the ultimate compressive strength. Two cements were tested. Biocement H was made of a powder containing alpha-tertiary calcium phosphate and precipitated hydroxyapatite. Biocement F was made of a powder containing, in addition, some monetite. The liquid/powder (L/P) ratio was varied over the range 0.30-0.40 ml g(-1), whereas the accelerator concentration in the liquid was varied from 0%-4% Na2HPO4 in water. For Biocement H there was no combination L/P ratio and % Na2HPO4 for which all clinical requirements were satisfied. However, Biocement F had a certain area where this was the case. Therefore, it is expected that Biocement F can be applied in clinical situations such as orthopaedics, plastic and reconstructive surgery and oral and maxillofacial surgery, even when early contact with blood is inevitable.

Some factors controlling the injectability of calcium phosphate bone cements.

The injectability of four calcium phosphate bone cements (CPBCs) was measured using a commercial disposable syringe. It varied considerably with the cement powder composition, with the liquid/powder ratio, with the time after starting the mixing of liquid and powder, with the accelerator concentration (% Na2HPO4), and with the ageing time of the cement powder which was prepared by milling. The injectability test could be used to determine accurately the dough time of CPBCs. Relations between the setting time and the cohesion time are discussed.